Name: the indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions
Uploaded: 2016-11-14T14:00:00
Description: Watch this Scientific Journal Video about The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions at JoVE.com

The present work was supported by the German research foundation (Deutsche Forschungsgemeinschaft DFG: GRK 736, Fa 159/22-1; the research school of the Ruhr University Bochum (GSC98/1) and the priority program SSP 1172 "Glia and Synapse", Fa 159/11-1,2,3).

The experiments with mice were in accordance with the German Law and the German Society for Neuroscience guidelines of animal husbandry. The animal care and utilization committees of the Ruhr-University Bochum have granted the appropriate permits.
1. Preparation and Cultivation of Cortical Astrocytes
Note: Complete these steps of the protocol at least 7 d before proceeding to the next steps, as the astrocyte cultures should develop into confluent monolayers before the...

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The main goal of the current protocol is to completely separate neuronal and astrocytic cultures, while maintaining them in shared medium. For this reason, the purity of the cultures obtained should be verified at the beginning of the procedure. We recommend the use of neuron-specific tubulin, neurofilaments or NeuN protein as neuronal markers, GFAP as astrocytic marker, O4 antigen as oligodendrocyte precursor marker and Iba1 protein to identify microglia.
Pay special attention when performing...

Throughout the past decades, the general interpretation of neuroglia function has evolved&#160;from the attribution of a merely supportive towards an active regulatory role concerning neuronal function1. Because of their prominent impact on brain homeostasis in health and disease2, astrocytes are of special interest for the scientific community. In the past few years, a diversity of studies have focused on neuron-glia interactions in vivo and in vitro3. However, most of the culture systems do not allow for the separate analysis of both cell types and of their respective secretomes.
Several approaches exploit the direct cocultivation of neurons and glia to achieve long lasting survival and physiologically relevant neuronal network development4-6. The present protocol reaches the same goals while keeping both cell types physically separated7. Compared to conditioned medium approaches8,9, our system allows to study the bidirectional communication between neurons and astrocytes. The expression of secreted signaling molecules can be monitored while the cells maturate in the shared medium. This opportunity is especially relevant, as astrocytes release soluble factors, such as cytokines, growth factors and extracellular matrix molecules10,11, thereby regulating neuronal growth and function7,12. Thus, it has been demonstrated that the addition of thrombospondin to retinal ganglion cells in vitro induces the formation of synapses13. However, other yet unknown factors are necessary to render synapses functional13. Furthermore, molecules released by astrocytes have to be identified in order to understand the basis of neuron-glia interactions.
The cultivation of primary neurons and astrocytes from mouse and rat has been described previously14-16. Here we present an elegant and versatile tool to combine both cell types in an indirect coculture approach. Since the two cultures are physically separated&#160;yet sharing the same medium, the impact of neurons, astrocytes and soluble molecules, can be separately analyzed, thus creating a powerful tool for neuron-glia interaction studies.

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			<td height="41" style="height:41px;width:115px;">Reagents</td>
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			<td height="63" style="height:63px;width:115px;">B27</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Cell culture grade water</td>
			<td style="width:152px;">MilliQ</td>
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			<td height="105" style="height:105px;width:115px;">Cytosine-&#223;-D arabinofuranoside (AraC)</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">C1768</td>
			<td style="width:191px;">CAUTION: H317, H361</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:115px;">DMEM</td>
			<td style="width:152px;">Gibco (Life Technologies)&#160;</td>
			<td style="width:151px;">41966-029</td>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">G1397</td>
			<td style="width:191px;">CAUTION: H317-334</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Glucose</td>
			<td style="width:152px;">Serva</td>
			<td style="width:151px;">22700</td>
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			<td height="63" style="height:63px;width:115px;">HBSS</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">14170-088</td>
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			<td height="63" style="height:63px;width:115px;">HEPES</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">15630-056</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:115px;">Horse serum</td>
			<td style="width:152px;">Biochrom AG</td>
			<td style="width:151px;">S9135</td>
			<td style="width:191px;"></td>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">C-2529</td>
			<td style="width:191px;"></td>
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			<td height="63" style="height:63px;width:115px;">MEM</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">A7641</td>
			<td style="width:191px;">CAUTION: H334</td>
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			<td height="42" style="height:42px;width:115px;">Papain</td>
			<td style="width:152px;">Worthington</td>
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			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">P0899</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:115px;">Poly-L-ornithine</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">P3655</td>
			<td style="width:191px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Sodium pyruvate</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td style="width:151px;">S8636</td>
			<td style="width:191px;"></td>
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			<td height="63" style="height:63px;width:115px;">Trypsin-EDTA</td>
			<td style="width:152px;">Gibco (Life Technologies)</td>
			<td style="width:151px;">25300054</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Equipment</td>
			<td style="width:152px;"></td>
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			<td height="42" style="height:42px;width:115px;">24 well plates</td>
			<td style="width:152px;">Thermoscientific/Nunc</td>
			<td style="width:151px;">142475</td>
			<td style="width:191px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:115px;">24-wells plate (for the&#160; indirect co-culture)</td>
			<td style="width:152px;">BD Falcon</td>
			<td style="width:151px;">353504</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:115px;">Binocular</td>
			<td style="width:152px;">Leica</td>
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			<td height="42" style="height:42px;width:115px;">Cell-culture inserts</td>
			<td style="width:152px;">BD Falcon</td>
			<td style="width:151px;">353095</td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:115px;">Centrifuge</td>
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			<td style="width:152px;">Marienfeld</td>
			<td style="width:151px;">650010</td>
			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:115px;">Forceps</td>
			<td style="width:152px;">FST Dumont (#5)</td>
			<td style="width:151px;">11254-20</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:115px;">glass cover slips (12 mm)</td>
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			<td style="width:151px;">P231.1</td>
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			<td height="42" style="height:42px;width:115px;">Micro tube (2 ml)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">72,691</td>
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			<td style="width:152px;">Millipore</td>
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			<td style="width:191px;"></td>
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			<td height="21" style="height:21px;width:115px;">Parafilm</td>
			<td style="width:152px;">Bemis</td>
			<td style="width:151px;">PM-996</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:115px;">Petri dishes (10 cm)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">833,902</td>
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			<td height="21" style="height:21px;width:115px;">pipette (1 ml)</td>
			<td style="width:152px;">Gilson</td>
			<td style="width:151px;">Pipetman 1000</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:115px;">Sterile work bench</td>
			<td style="width:152px;">The Baker Company</td>
			<td style="width:151px;">Laminar Flow SterilGARD III</td>
			<td style="width:191px;"></td>
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			<td height="42" style="height:42px;width:115px;">Surgical scissors</td>
			<td style="width:152px;">FST Dumont</td>
			<td style="width:151px;">14094-11</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Syringe</td>
			<td style="width:152px;">Henry Schein</td>
			<td style="width:151px;">9003016</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">T75 flask</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">833,911,002</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">tube (15 ml)</td>
			<td style="width:152px;">Sarstedt</td>
			<td style="width:151px;">64,554,502</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Water bath</td>
			<td style="width:152px;">GFL</td>
			<td style="width:151px;">Water bath type 1004</td>
			<td style="width:191px;"></td>
		</tr>
	</tbody>
</table>

the indirect neuron-astrocyte coculture assay: an in vitro set-up for the detailed investigation of neuron-glia interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The analysis of the neuronal cultures via the indirect coculture system is multifarious and can be performed at different stages of culture maturation. Due to the fact that the cells can be maintained for up to 4 weeks, long-term investigations of the cultures are possible.
The schematic in the middle left panel of Figure 1 demonstrates the cocultivation setup. With the use of this system, live cell imaging of b...

Watch this Scientific Journal Video about The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions at JoVE.com

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

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